BACKGROUND OF THE INVENTION:
[0001] The present invention relates to a novel pyrimidine nucleoside derivative and an
antiviral agent containing the derivative as active ingredient.
[0002] Infectious diseases caused by human acquired immunodeficiency virus (HIV), which
is a type of retrovirus, have recently become a serious social problem. A compound
of 3′-deoxy-3′-azidothymidine is known as a nucleoside compound used in the clinical
treatment for diseases caused by HIV-infection. However, this compound has side-effects
since it also exhibits considerable strong toxicity in the host cells.
[0003] Although some 2′,3′-dideoxyribonucleosides are known as nucleoside compounds exhibiting
an anti-retroviral activity, it is still necessary to develop a substance possessing
a higher activity and lower toxicity to the host cell (Hiroaki Mitsuya, Bodily Defence,
Vol. 4, pp.213-223 (1987)).
[0004] On the other hand, various acyclonucleoside compounds have been synthesized since
Acyclovir (acycloguanosine) was developed as an antiviral substance effective against
herpes virus (C.K. Chu and S.J. Culter, J. Heterocyclic Chem.,
23, p.289 (1986)). However, no compound having a sufficient activity especially against
retroviruses has yet been discovered.
[0005] Consequently, with the aim of providing an antiviral agent, especially an agent which
has an effective antiviral activity against retroviruses, the present inventors have
synthesized a wide variety of novel 6-substituted acyclopyrinudine nucleoside compounds
and their anti-retroviral activities have been investigated. As the results, the present
inventors have found that such an object can be attained by a specific 6-substituted
acyclopyrimidine nucleoside derivative (WO 89/09213).
[0006] As 6-substituted acyclopyrimidine nucleosides, 6-fluorine substituted derivatives
and 6-alkylamino substituted derivatives (DD-A-232492) and 6-methyl substituted derivatives
(C.A.
107, 129717w (1987)) are known, however, the antiviral activity of these compounds has
not been described.
[0007] The present inventors have conducted intensive studies on the screening of compounds
having antiviral activities, especially a compound having an anti-retroviral activity,
and found that among the 6-substituted acyclopyrimidine nucleoside derivatives generically
disclosed but not specifically disclosed in WO 89/09213, those pyrimidine nucleoside
derivatives having ethyl group or isopropyl group at the 5-position of the pyrimidine
ring and having (substituted) phenylthio group or (substituted) benzyl group showed
markedly excellent anti-retroviral activities. The present invention has been accomplished
based on this finding.
SUMMARY OF THE INVENTION:
[0008] The present invention intends to provide a pyrimidine nucleoside derivative represented
by the following formula (I):

wherein R¹ represents ethyl group or isopropyl group; R² and R³ independently represent
hydrogen atom, an C₁-C₃ alkyl group or chlorine atom; X represents oxygen atom or
sulfur atom; and Z represents sulfur atom or methylene group; with the proviso that
R² and R³ do not simultaneously represent hydrogen atoms when X represents oxygen
atom and Z represents sulfur atom.
[0009] The present invention intends to further provide an antiviral agent containing the
pyrimidine nucleoside derivative as active ingredient.
DETAILED DESCRIPTON OF THE INVENTION:
[0010] The pyrimidine nucleoside derivative of the present invention is represented by the
formula (I) shown above. In the formula (I), C₁-C₃ alkyl group as the substituents
R² and R³ may include methyl group, ethyl group, n-propyl group and isopropyl group.
[0012] The pyrimidine nucleoside derivative of the present invention can be synthesized
for instance in accordance with the following reaction scheme (1), (2) or (3).
(1) When Z in the formula (I) is sulfur atom.
(2) When Z in the formula (I) is a methylene group.

[0013] In the above reaction schemes, R¹, R², R³ and X represent the same as defined in
the formula (I), R⁴ represents a protection group for a hydroxyl group and M represents
an alkali metal.
[0014] The protecting group expressed by R⁴ in the above reaction schemes may be selected
from protecting groups usually used for protecting alcohol, provided that they do
not undergo elimination under alkaline conditions.
[0015] Illustrative examples of such protecting groups may include aralkyl groups such as
benzyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl and the like;
silyl groups such as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,
dimethylphenylsilyl and the like; tetrahydropyranyl; and substituted alkyl groups
such as methoxymethyl and the like. Of these, silyl groups may be most preferable.
[0016] As the first step of the synthesis of the pyrimidine nucleoside derivative of the
present invention, a compound represented by the above general formula (II) or (VI)
is allowed to react with an organic alkali metal compound at a temperature of from
-80°C to -10°C for 0.2 to 10 hours in a solvent, for example, an ether solvent such
as diethyl ether, tetrahydrofuran and the like. The organic alkali metal compound
may include potassium bistrimethylsilylamide, sodium bistrimethylsilylamide and lithium
alkylamides. Of these, lithium diisopropyl amide (LDA) and lithium 2,2,6,6-tetramethylpiperidide
(LTMP ) may be most preferable. A lithium alkylamide may preferably be prepared just
before its use in the reaction system. For example, a preferable lithium alkylamide
may be prepared by allowing a secondary amine such as diisopropylamine to react with
an alkyl lithium for instance n-butyl lithium, at a temperature of from -80°C to -10°C
for 0.2 to 5 hours with stirring in a solvent such as diethyl ether, dioxane, tetrahydrofuran,
dimethoxyethane or the like under an atmosphere of inert gas such as argon gas.
[0017] The organic alkali metal compound may generally be used in an amount of from 2 to
5 moles per one mole of a compound represented by the general formula (II) or (VI).
[0018] Next, about 1 to 5 moles of a thioarylating agent, (substituted) benzaldehyde or
an alkylating agent is added to one mole of a compound represented by the general
formula (II) or (VI), and these compounds are allowed to react each other under similar
conditions to the reaction of the organic alkali metal compound. The thioarylating
agent may include diphenyl disulfide, a benzenesulfenyl halide and the like, each
of which may have at least one substituent of R² and R³.
[0019] Illustrative examples of the alkylating agent may include alkyl halides such as ethyl
chloride, isopropyl chloride, ethyl bromide, isopropyl bromide, ethyl iodide, isopropyl
iodide and the like; and alkyl sulfonates such as ethyl mesylate, isopropyl mesylate,
ethyl tosylate, isopropyl tosylate and the like.
[0020] The compound represented by the general formula (II) as a starting material may be
prepared by known methods. For example, such a starting compound of the formula (II)
can be obtained by carrying out condensation reaction of a trimethylsilylated uracil
derivative with (2-acetoxyethoxy)methylbromide, hydrolyzing the condensed product
and then introducing the aforementioned protecting group. Such preparation processes
have been disclosed in detail for instance in Can. J. Chem., vol.
60, 547(1982).
[0021] Introduction of a protecting group can be carried out in the usual way. For example,
a silyl protecting group can be introduced by allowing a compound to react with 1
to 10 times by mole of a silylating agent such as trimethylsilyl chloride, t-butyldimethylsilyl
chloride or the like in a solvent such as dimethylformamide, acetonitrile, tetrahydrofuran
or the like or in a mixture thereof, at a reaction temperature of from 0 to 50°C in
the presence of a base such as pyridine, picoline, diethylaniline, dimethylaniline,
triethylamine, imidazole or the like. The compound represented by the general formula(VI)
can be synthesized in similar manner to the process of the reaction scheme (1).
[0022] Prior to the elimination of a protecting group, the thus prepared compounds represented
by the general formulae (III), (IV) and (VII) are subjected, if necessary, to separation
and purification steps usually used for the separation and purification of nucleosides,
such as recrystallization, adsorption chromatography, ion exchange chromatography
and the like.
[0023] Elimination of the protecting group may be carried out by selecting a suitable method
from usually used methods such as acid hydrolysis, ammonium fluoride treatment, catalytic
reduction and the like, depending on the protecting group to be removed.
[0024] The compound represented by the general formula (V) which has been derived from the
compound (IV) according to the reaction scheme (2) is further subjected to a reduction
step to obtain the pyrimidine nucleoside derivative of the present invention represented
by the general formula (I). Reduction of the compound (V) may be effected for instance
by using hydrogen in the presence of palladium carbon, palladium hydroxide or the
like.
[0025] The thus prepared pyrimidine nucleoside derivative of the present invention represented
by the general formula (I) can be separated and purified by a method usually used
for the separation and purification of nucleosides, such as recrystallization and
adsorption or ion exchange chromatography.
[0026] The pyrimidine nucleoside derivative of the present invention may be made into a
pharmaceutically acceptable salt by conventional methods. Examples of such salts may
include alkali metal salts such as sodium salt, potassium salt and the like; alkaline
earth metal salts such as magnesium salt and the like; and ammonium salts such as
ammonium salt, methylammonium salt, dimethylammonium salt, trimethylammonium salt,
tetramethylammonium salt and the like.
[0027] The pyrimidine nucleoside derivative of the present invention can be administered
to a patient through any of the usual routes such as oral, rectal, parenteral and
local administrations for the purpose of preventing infection of retroviruses and
the like or treating infectious diseases caused by these viruses. Though it must be
decided depending on the age, physical condition, body weight and the like of each
patient, appropriate administration dose of the derivative of the present invention
may be generally in the range of from 1 to 100 mg/kg(body weight)/day, preferably
from 5 to 50 mg/kg(body weight)/day. Administration of the derivative of the present
invention may be made once a day or a few times a day within the above range of dose.
[0028] For the purpose of formulating pharmaceutical preparations, the derivative of the
present invention may be made into a composition containing usually used carriers,
excipients and other additive agents. The carriers may be in either a solid or a liquid
form. Illustrative examples of solid carriers may include lactose, china clay (kaolin),
sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium
stearate, lecithin, sodium chloride and the like. Illustrative examples of liquid
carried may include glycerin, peanut oil, polyvinyl pryrrolidone, olive oil, ethanol,
benzyl alcohol, propylene glycol, water and the like.
[0029] The antiviral agent of the present invention can be made into various forms. When
solid carriers are used, for example, the antiviral agent can be made into tablet,
powder, granule, capsule, suppository, troche and the like. When liquid carriers are
used, it can be made into syrup, emulsion, soft gelatin capsule, cream, gel, paste,
spray and the like, as well as injection solution.
[0030] The present invention will be further illustrated hereinafter referring to the following
non-limitative Examples.
Example 1:
[0031] Preparation of 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-phenylthio-2-thiouracil (compound
No. 1 in Table 1)
[0032] To 6.2 g (40 mmol) of 5-ethyl-2-thiouracil suspended in 100 ml of dichloromethane
was added 22 ml (88 mmol) of bis-(trimethylsilyl)-acetamide under a nitrogen atmosphere
at room temperature, and the mixture was stirred for 3 hours. To this was further
added gently 3.4 ml (48 mmol) of 1,3-dioxolan and 5.6 ml (48 mmol) of tin tetrachloride.
The resulting mixture was then subjected to reflux for 17 hours. The reaction mixture
thus obtained was poured into 100 ml of a mixture of methanol and water (1:1) containing
22 g of sodium bicarbonate. After stirring for 2 hours, the resulting mixture was
filtered through sellaite and the filtrate was evaporated to dryness. To the residue
was added 120 ml of acetonitrile, 12 g (80 mmol) of t-butyldimethylsilyl chloride
and 5.4 g (80 mmol) of imidazole under a nitrogen atmosphere at room temperature.
After 14 hours of stirring, the resulting reaction mixture was concentrated and subjected
to partition using an ethyl acetate-water system, and then the organic layer was evaporated
to dryness. The residue was adsorbed on a silica gel columm and eluted with chloroform.
Thereafter, the eluent was subjected to crystallization from a chloroform-hexane solvent
to obtain 7.2 g (52%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyl-2-thiouracil.
[0033] Next, 11 ml (22 mmol) of 2.0 M solution of lithium diisopropylamide in tetrahydrofuran
was added under a nitrogen atmosphere to 30 ml of tetrahydrofuran which has been cooled
down to -70°C in advance. To this was added dropwise 14 ml of tetrahydrofuran solution
containing 3.4 g (10 mmol) of the thus obtained 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyl-2-thiouracil
by keeping the reaction solution at a temperature of -70°C or lower. The resulting
mixture was stirred for 1 hour at -70°C. To this was added dropwise 10 ml of tetrahydrofuran
solution containing 2.8 g (13 mmol) of diphenyl disulfide. After 1 hour, the resulting
reaction mixture was further mixed with 1.3 ml of acetic acid, adjusted to room temperature
and subjected to partition using an ethyl acetate-water system, and then the organic
layer was evaporated to dryness. Thereafter, the residue was adsorbed on a silica
gel column and eluted with chloroform to obtain 3.4 g (76%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyl-6-phenylthio-2-thiouracil.
[0034] A 0.42 g (0.94 mmol) portion of the thus obtained compound was dissolved in 5 ml
of methanol, and the solution was adjusted to pH1 with 1 N HCl. Thereafter, the solution
was evaporated to dryness and then subjected to crystallization from an ethyl acetate-hexane
solvent to obtain 0.19 g (60%) of the titled compound. The melting point of the obtained
compound was determined to be 71 to 75°C.
Example 2:
[0035] Preparation of 6-(3,5-dimethylphenylthio)-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil
(compound No. 2 in Table 1)
[0036] The titled compound, having the melting point of 121 to 125°C, was obtained by repeating
the process of Example 1 except that 5-ethyluracil was used instead of 5-ethyl-2-thiouracil
and that 3,3′,5,5′-tetramethyldiphenyl disulfide was used instead of diphenyl disulfide.
Example 3:
[0037] Preparation of 6-(3,5-dimethylphenylthio)-5-ethyl-1-[(2-hydroxyethoxy)methyl]-2-thiouracil
(compound No. 3 in Table 1)
[0038] The titled compound, having the melting point of 121 to 123°C, was obtained by repeating
the process of Example 1 except that diphenyl disulfide was replaced by 3,3′,5.5′-
tetramethyldiphenyl disulfide.
Example 4:
[0039] Preparation of 6-(3,5-dichlorophenylthio)-5-ethyl-1-[(2-hydroxyethoxy)methyl] uracil
(compound No. 6 in Table 1)
[0040] The titled compound, having the melting point of 93 to 95°C, was obtained by repeating
the process of Example 1 except that 5-ethyluracil was used instead of 5-ethyl-2-thiouracil
and that 3,3′,5,5′-tetrachlorodiphenyl disulfide was used instead of diphenyl disulfide.
Example 5:
[0041] Preparation of 6-(3,5-dichlorophenylthio)-5-ethyl-1-[(2-hydroxyethoxy)methyl]-2-thiouracil
(compound No. 7 in Table 1)
[0042] The titled compound, having the melting point of 91 to 93°C, was obtained by repeating
the process of Example 1 except that diphenyl disulfide was replaced by 3,3′,5,5′-
tetrachlorodiphenyl disulfide.
Example 6:
[0043] Preparation of 6-benzyl-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil (compound No. 10
in Table 1)
[0044] To 5.6 g (40 mmol) of 5-ethyluracil suspended in 100 ml of dichloromethane was added
22 ml (88 mmol) of bis-(trimethylsilyl)-acetamide under a nitrogen atmosphere at room
temperature, and the mixture was stirred for 3 hours. To this was further added gently
3.4 ml (48 mmol) of 1,3-dioxolan and 5.6 ml (48 mmol) of tin tetrachloride. The resulting
mixture was then subjected to reflux for 17 hours. The reaction mixture thus obtained
was poured into 100 ml of a mixture of methanol and water (1:1) containing 22 g of
sodium bicarbonate. After stirring for 2 hours, the resulting mixture was filtered
through sellaite and the filtrate was evaporated to dryness. To the residue was added
120 ml of acetonitrile, 12 g (80 mmol) of t-butyldimethylsilylchloride and 5.4 g (80
mmol) of imidazole under a nitrogen atmosphere at room temperature. After 14 hours
of stirring, the resulting reaction mixture was concentrated and subjected to partition
using an ethyl acetate-water system, and then the organic layer was evaporated to
dryness. The residue was adsorbed on a silica gel column and eluted with chloroform.
Thereafter, the eluent was subjected to crystallization from a chloroform-hexane solvent
to obtain 6.9 g (52%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyluracil.
[0045] Next, 11 ml (22 mmol) of 2.0 M solution of lithium diisopropylamide in tetrahydrofuran
was added under a nitrogen atmosphere to 30 ml of tetrahydrofuran which has been cooled
down to -70°C in advance. To this was added dropwise 14 ml of tetrahydrofuran solution
containing 3.3 g (10 mmol) of the thus obtained 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyluracil
by keeping the reaction solution at a temperature of -70°C or lower. The resulting
mixture was stirred for 1 hour at -70°C. To this was dropwisely added 10 ml of tetrahydrofuran
solution containing 1.3 ml (13 mmol) of benzaldehyde. After 1 hour, the resulting
reaction mixture was further mixed with 1.3 ml of acetic acid, adjusted to room temperature
and subjected to partition using an ethyl acetate-water system, and then the organic
layer was evaporated to dryness. Thereafter, the residue was adsorbed on a silica
gel column and eluted with chloroform to obtain 2.7 g (61%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyl-6-[(1-hydroxy-1-phenyl)-methyl]uracil.
[0046] A 2.7 g (6.1 mmol) portion of the thus obtained compound was dissolved in 15 ml of
methanol. The resulting solution was adjusted to pH1 with 1 N HCl, allowed to stand
for 1 hour at room temperature and when neutralized with a sodium hydroxide solution.
Thereafter, the thus neutralized solution was evaporated to dryness and then subjected
to crystallization from an ethyl acetate-hexane solvent to obtain 1.8 g (90%) of 5-ethyl-1-[(hydroxyethoxy)methyl]-6-[(1-hydroxy-1-phenyl)methyl]uracil.
[0047] A 1.8 g (5.5 mmol) portion of the thus obtained compound was dissolved in 75 ml of
ethanol. The resulting solution was added with 0.2 g of a 20% palladium hydroxide-carbon
catalyst and the mixture was stirred at 60°C for 14 hours under a hydrogen atmosphere.
The catalyst in the reaction mixture was removed by filtration. Thereafter, the filtrate
was evaporated to dryness and then subjected to crystallization from an ethyl acetate-hexane
solvent to obtain 1.6 g (93%) of the titled compound. The melting point of the obtained
compound was determined to be 121 to 121.5°C.
Example 7:
[0048] Preparation of 6-(3,5-dimethylbenzyl)-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil (Compound
No. 12 in Table 1)
[0049] The titled compound, having the melting point of 175 to 177°C, was obtained by repeating
the process of Example 6 except that 3,5-dimethylbenzaldehyde was used instead of
benzaldehyde.
Example 8:
[0050] Preparation of 1-[(2-hydroxyethoxy)methyl]-6-phenylthio-5-isopropyl-2-thiouracil
(Compound No. 20 in Table 1)
[0051] The titled compound, having the melting point of 145 to 147°C, was obtained by repeating
the process of Example 1 except that 5-isopropyl-2-thiouracil was used instead of
5-ethyl-2-thiouracil.
Example 9:
[0052] Preparation of 6-(3,5-dimethylphenylthio)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil
(Compound No. 21 in Table 1)
[0053] The titled compound, having the melting point of 138 to 139°C, was obtained by repeating
the process of Example 1 except that 5-isopropyluracil was used instead of 5-ethyl-2-thiouracil
and that 3,3′,5,5′-tetramethyldiphenyl disulfide was used instead of diphenyl disulfide.
Example 10:
[0054] Preparation of 6-(3,5-dimethylphenylthio)-1-[(2-hydroxyethoxy)methyl]-5-isopropyl-2-thiouracil
(Compound No. 22 in Table 1)
[0055] The titled compound, having the melting point of 140 to 141°C, was obtained by repeating
the process of Example 1 except that 5-isopropyl-2-thiouracil was used instead of
5-ethy-2-thiouracil and that 3,3′,5,5′-tetramethyldiphenyl disulfide was used instead
of diphenyl disulfide.
Example 11:
[0056] Preparation of 6-(3,5-dimethylbenzyl)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil
(Compound No. 31 in Table 1)
[0057] The titled compound, having the melting point of 187.5 to 188.5°C, was obtained by
repeating the process of Example 6 except that 5-isopropyluracil was used instead
of 5-ethyluracil and that 3,3′,5,5′-tetramethylbenzaldehyde was used instead of benzaldehyde.
Synthetic Example 1:
[0058] Preparation of 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)uracil (compound
No. 39)
[0059] To 5.6 g (40 mmol) of 5-ethyluracil suspended in 100 ml of dichloromethane was added
22 ml (88 mmol) of bis-(trimethylsilyl)acetamide under a nitrogen atmosphere at room
temperature, and the mixture was stirred for 3 hours. To this was further added gently
3.4 ml (48 mmol) of 1,3-dioxolan and 5.6 ml (48 mmol) of tin tetrachloride. The resulting
mixture was then subjected to reflux for 17 hours. The reaction mixture thus obtained
was poured into 100 ml of a mixture of methanol and water (1:1) containing 22 g of
sodium bicarbonate. After stirring for 2 hours, the resulting mixture was filtered
through sellaite and the filtrate was evaporated to dryness. To the residue was added
120 ml of acetonitrile, 12 g (80 mmol) of t-butyldimethylsilyl chloride and 5.4 g
(80 mmol) of imidazole under a nitrogen atmosphere at room temperature. After 14 hours
of stirring, the resulting reaction mixture was concentrated and subjected to partition
using an ethyl acetate-water system, and then the organic layer was evaporated to
dryness. The residue was adsorbed on a silica gel column and eluted with chloroform.
Thereafter, the eluent was subjected to crystallization from a chloroform-hexane solvent
to obtain 6.9 g (52%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyluracil.
[0060] Next, 11 ml (22 mmol) of 2.0 M solution of lithium diisopropylamide in tetrahydrofuran
was added under a nitrogen atmosphere to 30 ml of tetrahydrofuran which has been cooled
down to -70°C in advance. To this was added dropwise 14 ml of tetrahydrofuran solution
containing 3.3 g (10 mmol) of the thus obtained 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyluracil
by keeping the reaction solution at a temperature of -70°C or lower. The resulting
mixture was stirred for 1 hour at -70°C. To this was dropwisely added 10 ml of tetrahydrofuran
solution containing 2.8 g (13 mmol) of diphenyl disulfide. After 1 hour, the resulting
reaction mixture was further mixed with 1.3 ml of acetic acid, adjusted to room temperature
and subjected to partition using an ethyl acetate-water system, and then the organic
layer was evaporated to dryness. Thereafter, the residue was adsorbed on a silica
gel column and eluted with chloroform to obtain 3.4 g (76%) of 1-[(2-t-butyldimethylsilyloxyethoxy)methyl]-5-ethyl-6-(phenylthio)uracil.
[0061] A 0.41 g (0.94 mmol) portion of the thus obtained compound was dissolved in 5 ml
of methanol, and the solution was adjusted to pH 1 with 1 N HCl. Thereafter, the solution
was evaporated to dryness and then subjected to crystallization from an ethyl acetate-hexane
solvent to obtain 0.18 g (60%) of the titled compound. The melting point of the obtained
compound was determined to be 117 to 120°C.
Synthetic Example 2:
[0062] Preparation of 1-[(2-hydroxyethoxy)methyl]-6-phenylthio-5-isopropyluracil (compound
No. 40)
[0063] The titled compound, having the melting point of 85 to 87°C, was obtained by repeating
the process of Synthetic Example 1 except that 5-isopropyluracil was used in place
of 5-ethyluracil.
Example 12: Preparation of tablet
[0064]

[0065] The above components where well mixed and tablets were produced by a direct tableting
method. Each tablet thus prepared had a weight of 100 mg and contained 1.0 mg of 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-phenylthio-2-thiouracil.
Example 13: Preparation of powder and capsule
[0066]

[0067] Both powder components were well mixed to obtain a powder formulation. Capsule was
obtained by encapsulating 100 mg of the thus obtained powder into a hard capsule of
No. 5.
Example 14: Preparation of tablet
[0068]

[0069] The above components were well mixed and tablets were produced by a direct tableting
method. Each tablet thus prepared had a weight of 100 mg and contained 1.0 mg of 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)uracil.
Example 15: Preparation of powder and capsule
[0070]

[0071] Both powder components were well mixed to obtain a powder formulation. Capsule was
obtained by encapsulating 100 mg of the thus obtained powder into a hard capsule of
No. 5.
Example 16: Inhibitory activity for HIV infection
[0072] In RPMI 1640 DM culture medium containing 20 mM of Hepes buffer solution, 10 % fetal
bovine serum and 20 g/ml of gentamycin, 3 X 10⁴ MT-4 cells (human T cell clone which
is destroyed by the infection of HIV) were infected with HIV in an amount of 100 times
as large as expected to cause 50 % infection of the cells. Immediately thereafter,
a predetermined amount of sample was added to the culture medium using 50 mg/ml sample
solutions in dimethyl sulfoxide and the cells were cultured at 37°C.
[0073] After 5 days of incubation, the number of existing cells was counted to determine
the concentration of the compound required for preventing the death of 50 % of the
MT-4 cells. Separately, MT-4 cells were cultured in the same way as above except that
they were not infected with HIV to determine the concentration of the compound at
which 50 % of the MT-4 cells were destroyed.
[0074] The result are shown in Table 2 (compound numbers in Table 2 correspond to those
in Table 1 and Examples).
